AG Daniel:Tools

AG Daniel

Tools

BD FACScan Flowcytometer

The FACScan has one laser at 488 nm and can collect 3 flourescent and 2 scatter properties simultaneously. It is important to remember that only fluorochromes excited at ~488nm may be used on this instrument.

Laser	Parameter	Filter	Common Dyes/Fluorochromes
488 nm	FL-1	530/30	FITC, GFP, SYBR Green-I
	FL-2	585/42	Phycoerythrin, PI
	FL-3	670LP	Chlorophyll, CyChrome

Quickguide

1. TURN ON FACSCAN MACHINE FIRST THEN COMPUTER.
2. CLEAR FLOW CELL OF TRAPPED AIR BUBBLES BY DRAINING AND FILLING:

     1. Open the optics compartment door, above fluidics compartment.
     2. While viewing the flow cell (quartz triangular cuvette on lower, right side), 
        turn the fluid control to "DRAIN" until you see that all the sheath fluid has 
        drained from the flow cell.
     3. Next, turn the fluid control dial to "FILL" and watch as the flow cell steadily 
        fills with sheath fluid.
     4. Repeat "DRAIN" and "FILL" 2-3 times, until no bubbles are visible upon filling.
     5. Turn fluid control to "STANDBY".
           NOTE: "BACKFLUSH" reverses the flow of sheath to flush fluid out of the sample 
           injection tube to help remove clogs. Ensure a tube of water and not your sample 
           is in the sample injection port at the time and follow "BACKFLUSH" with "DRAIN", 
           then "FILL" to refill the flow cell.  

3. LAUNCH CELLQUEST PRO SOFTWARE FROM THE DOCK ICON.
4. OPEN FILE AND UNDER THE "ACQUIRE" TOOL BAR,OPEN THE FOLLOWING FROM THE DROP DOWN MENU:

     1. "CONNECT TO CYTOMETER": Automatically opens "ACQUISITION CONTROL WINDOW"
     2. "ACQUISITION": To identify what data is stored and how many events (cells) to collect.
     3. "PARAMETER": To identify where data is stored and how each sample can be individually 
        labeled.
         1. Click on "FOLDER" and locate your folder (or create your folder) 
         2. Click on "FILE", then "CUSTOM PREFIX", select "SAMPLE ID": This allows you to 
            enter a sample description or name in the SAMPLE ID block.
         3. "COUNTER": To show the status of sample collection and number of collected events.

5. UNDER THE "CYTOMETER" TOOL BAR, OPEN THE FOLLOWING FROM THE DROP DOWN MENU:

     1. "DETECTOR/AMPS": To select the parameters for your fluorochrome (discussed later).
     2. Optional to open, "THRESHOLD": To establish the baseline level of detection.
     3. Optional to open, "COMPENSATION": Used when two fluorochromes are used to separate the 
        overlaps in wavelength emission.
     4. Optional to open, "STATUS": Provides status of laser, fluid levels and if at "READY".
     5. "INSTRUMENT SETTINGS": Allows you to save your optimized detector/amp settings or to 
        retrieve saved settings in your own folder/Zip disk, thus saving time.

6. TO CREATE A NEW MASK

   UNDER THE "PLOT" TOOL BAR, OPEN THE FOLLOWING FROM THE DROP DOWN MENU TO CREATE A:
     1. "HISTOGRAM":
         1. "PLOT SOURCE", choose "ACQUISITION" to collect data or "ANALYSIS" to view collected 
            data files.
         2. "PARAMETER", choose the appropriate one for your fluorochrome.
     2. "DOTPLOT":
         1. "PLOT SOURCE", choose "ACQUISITION" to collect data or "ANALYSIS" to view collected 
            data files.
         2. "X-PARAMETER", choose the appropriate one for your fluorochrome.
         3. "Y-PARAMETER", choose the appropriate one.

7. TO COLLECT DATA, LOAD SAMPLE:

     1. Your are ready to collect data when the "READY" light on FACScan control panel is lit.
     2. Turn fluid control to "RUN"
     3. Remember, use only FALCON polystyrene, 12 x 75mm tubes, #352054 which fits properly on
        the sample injection port.
     4. Turn the tube support arm to the right, and install sample tube on sample injection port, 
        then quickly center the tube support arm under the tube.

8. CLICK "ACQUIRE" IN THE ACQUISITION CONTROL WINDOW:

     1. While the "SETUP" box is checked, you may view live data but the data will not be saved. 
        This setup mode is simply used to allow optimization of settings.
     2. When you are finished with step 9, you should click the setup box (which removes the "X") 
        so that you can save sample data to files.

9. OPTIMIZE SETTINGS IN THE DETECTORS/AMPS WINDOW:

     1. If you are acquiring sample for DNA analysis, click the "DDM" box then select the DDM 
        parameter for your fluorochrome.
     2. Adjust the detector levels by moving the "VOLTAGE" to get the population on scale.
     3. Adjust the "GAIN" to allow you the choice of processing each of the signals through a log 
        or linear amplifier depending on your application.
     4. In general, for common fluorochromes adjust the voltage for the parameters:
         1. SSC and FSC parameters: Used for FITC cell surface staining visualization.
         2. FL1 parameter: Used for GFP visualization.
         3. FL2 parameter: Used for Propidium Iodide staining for DNA cell cycle/apoptosis analysis.

10. SHUTDOWN PROCEDURE:

     1. Run FACS Clean in sample injection port for 5-10 minutes.
         1. Do not submerge black connection at top of sample port.
     2. Run FACS Save in sample injection port for 5-10 minutes.
     3. Run ddwater in sample injection port for 5 minutes.
     4. Leave ddwater in sample port.
     5. Turn to "STANDBY"

11. TURN OFF FACSCAN AND SHUTDOWN COMPUTER.

A. CHANGE SHEATH RESERVOIR:

     1. Unscrew cap and remove sheath reservoir.
     2. Use empty SHEATH as new WASTE
     3. Open new SHEATH, screw cap on.
     4. Press ALARM to stop beeping.

B. EMPTY WASTE RESERVOIR:

     1. Remove waste reservoir; unscrew cap; and empty down drain.
     2. Press ALARM to stop beeping.

Materials

Dynabeads

Dynabeads® are superparamagnetic, monosized polymer particles, providing a solid-phase with liquid-phase kinetics. A wide range of bioreactive molecules can be adsorbed or coupled to the bead-surface, and used in the separation of biological materials (cells, proteins, nucleic acids etc). Dynabeads

Name	Cat. number	storage	comment
CD4 (T helper/inducer)		Bernd (upstairs)	old stuff
CD14		Bernd	old stuff
CD19 (panB)		Bernd	old stuff
CD4 Positive Isolation Kit		Bernd	old stuff
CD8 Positive Isolation Kit		Bernd	old stuff
T Cell Negative Isolation Kit		Bernd	old stuff
CELLection Kit		Bernd	old stuff

ELISA

Bender Medsystems

Name	Cat. number	storage	comment
human FasL ELISA		fridge 0402	old stuff
human IFN-gamma		fridge 0402	old stuff

MACS

MACS® Technology is based on MACS MicroBeads, manual or automated MACS Separators, and MACS Columns. When MACS Columns are placed in a MACS Separator, the MACS Column matrix provides a magnetic field strong enough to retain cells labeled with minimal amounts of magnetic material. Therefore, MACS MicroBeads used for labeling the cells can be so small and only a few are needed to separate a cell.

The magnetically labeled cells are separated over a MACS Column placed in a MACS Separator. They are retained on the column, while unlabeled cells pass through. These cells can be collected as the unlabeled fraction. The retained cells are eluted from the MACS Column after removal from the magnet. MACS

Name	Cat. number	storage	comment
Apoptotic Cell Isolation Kit		Bernd (upstairs)	old stuff
Goat anti-Mouse IgG Microbeads		Bernd	old stuff
CD19 (panB)		Bernd	old stuff
CD14 Microbeads		Bernd	old stuff